Inflammation and Syndecan-4 Shedding from Cardiac Cells in Ischemic and Non-Ischemic Heart Disease

Figures

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  Figure 1. Syndecan-4 is shed in and from the human heart. (A) Schematic illustrating the antibody used to detect full-length (FL) syndecan-4 and the cellular fragment (CF) left in cells after shedding of the ectodomain, by immunoblotting of myocardial tissue or cellular protein lysates (left panel), and the antibody detecting the shed ectodomain (SE) in blood (right panel) by enzyme-linked immunosorbent assay (ELISA). (B) Representative immunoblot of human FL (upper arrowhead) syndecan-4 and the CF (lower arrowhead) in myectomy septum biopsies from patients with hypertrophic obstructive cardiomyopathy, n = 3. (C) Serum syndecan-4 levels (ng/mL) in peripheral blood from the radial artery and blood from the coronary sinus of aortic stenosis patients obtained during open heart surgery, n = 15. The numerical data supporting the graph can be found in Supplementary Table S1. (D) Schematic illustrating the adenoviral vectors used to transduce cultures of primary neonatal rat cardiac cells. Cells were transduced with an Adenovirus serotype 5 (dE1/E3) with RGD fiber modification encoding mouse syndecan-4 (AdSdc4) or vehicle (AdNull). (E,F) Representative immunoblots of mouse syndecan-4 FL and CF in…

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  Figure 2. Flow diagrams of included patients. (A) Flow diagram of cardiomyopathy patients included from the Maastricht Dilated Cardiomyopathy Registry. (B) Flow diagram of patients with acute myocardial infarction (MI) enrolled at University Hospitals Leuven.

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  Figure 3. Serum syndecan-4 levels do not reflect the inflammatory status of the heart in patients with heart disease. Serum samples were collected from patients with cardiomyopathy of various etiologies. Patients were extensively characterized. Serum syndecan-4 levels (ng/mL) were measured by enzyme-linked immunosorbent assay (ELISA) by one experienced researcher blinded to patient characteristics. The cohort (n = 430) consisted of subgroups with specific diagnoses, i.e., inflammatory (n = 71) and non-inflammatory (n = 318) dilated cardiomyopathy (DCM), myocarditis (n = 15), pericarditis (n = 3) and perimyocarditis (n = 23). Serum samples were obtained from n = 119 patients with acute myocardial infarction (MI), with or without ST-elevation, at day of admission (day 0), day 3 day and day 30 post-MI. We included serum samples from n = 5 healthy controls. Data in the graph are presented as individual data points and mean ± S.D for patients (red) and controls (blue). The numerical data supporting the graph can be found in Supplementary Table S2. Descriptive statistics are included underneath the graph. Statistical differences were tested using ordinary or repeated measures one-way ANOVA with…

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  Figure 4. Attenuation of interleukin 1β-increased syndecan-4 levels immunomodulatory therapy in cultured cardiomyocytes. Primary cardiomyocyte cultures were prepared from neonatal rat hearts. (A) Relative mRNA expression of endogenous rat syndecan-4 (Sdc4) in cardiomyocytes following 24 h treatment with interleukin (IL)-1β (10 ng/mL) compared to non-treated control or co-treatment with the IL-1 receptor antagonist (IL-1Ra; 100 ng/mL), n = 6. (B) Representative immunoblot and quantification of full-length (FL) syndecan-4 and the cellular fragment (CF) seen in cells after release of the ectodomain, in cardiomyocytes transduced with an adenovirus encoding mouse syndecan-4 (AdSdc4) and after 24 h treatment with IL-1β compared to non-treated or co-treatment with IL-1Ra, n = 9. (C) Relative mRNA levels of endogenous rat syndecan-4 in cardiomyocytes after 24 h treatment with tumor necrosis factor (TNF)α (10 ng/mL) compared to non-treated control or co-treatment with the anti-TNFα antibody infliximab (100 ng/mL), n = 6. (D) Representative immunoblot and quantification of syndecan-4 FL and CF in cardiomyocytes transduced with mouse AdSdc4 and treated for 24 h with TNFα vs. non-treated or…

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  Figure 5. Interleukin 1β-increased syndecan-4 levels and shedding are attenuated by immunomodulatory therapy in cultured cardiac fibroblasts. Primary cardiac fibroblast cultures were prepared form neonatal rat hearts. (A) Relative mRNA levels of rat syndecan-4 (Sdc4) in cardiac fibroblasts after 24 h treatment with interleukin (IL)-1β (10 ng/mL) compared to non-treated control or co-treatment with the IL-1 receptor antagonist (IL-1Ra; 100 ng/mL), n = 6. (B) Representative immunoblot and quantification of full-length (FL) syndecan-4 and the cellular fragment (CF) remaining after shedding of the ectodomain, in rat cardiac fibroblasts transduced with an adenovirus encoding mouse syndecan-4 (AdSdc4) and following 24 h treatment with IL-1β compared to non-treated or co-treatment with IL-1Ra, n = 12. (C) Relative mRNA of rat syndecan-4 in cardiac fibroblasts after 24 h treatment with tumor necrosis factor (TNF)α (10 ng/mL) vs. non-treated control or co-treatment with the anti-TNFα antibody infliximab (100 ng/mL), n = 6. (D) Representative immunoblot and quantification of syndecan-4 FL and CF in cardiac fibroblasts transduced with mouse AdSdc4 and treated for 24 h with TNFα vs. non-treated or…